Developments in commercial and academic medical and biotechnology have driven a strong focus on methods for biological cell sorting. The two main approaches that have emerged—bulk separation and single cell sorting—both enrich a population of cells with a targeted subset with specific physicochemical (i.e. size, volume, light scattering properties, etc.), immunological, or functional characteristics. Bulk sorting generally focuses upon a single discriminating cellular feature. Examples include cell filtration, centrifugation/sedimentation and affinity-based panning methods. The main disadvantages of bulk sorting are lower purity, loss of cells during the sorting process, difficulty in sorting out relatively rare cells, and difficulty in discriminating among similar sub-populations of cells. Bulk sorting, however, is a relatively simple method that offers high throughput. In contrast, single cell methods, the most important of which is fluorescence activated cell sorting (FACS) by flow cytometry, examine each cell individually to target the desired subpopulation for isolation and then guide them into different output streams. The reduction in throughput is offset by major advantages in specificity of sorting that is tunable to the desired outcome, generally higher recovery of cells, the ability to sort rare or only weakly discriminated cell populations, and the availability of multi-target sorting based on an array of multiple cellular features (i.e. several types of surface receptor, each tagged with a different fluorescent label). One important challenge faced by FACS flow cytometric methods is the damage incurred by some cells in the flow (shear stress) and sorting (electric field damage) processes. An important example is the reduced fertility of sorted sperm samples that can be attributed to these disruptive physical processes.
In the agriculture sector, cell discrimination is particularly important in livestock species where artificial insemination is commonly practised such as cattle. The use of sexed semen facilitates control of offspring gender for commercial benefit. The current commercially important method for sperm sorting uses FACS flow cytometry, in which sperm are discriminated by their differences in DNA content. The DNA of each spermatazoon is stained with a fluorescent dye in proportion to the DNA content. As the X chromosome is larger (i.e. has more DNA) than the Y chromosome, the “female” (X-chromosome bearing) spermatozoa will absorb a greater amount of dye than the “male” (Y-chromosome bearing) spermatozoa and as a consequence when exposed to UV light during flow cytometry will fluoresce with higher intensity than the Y spermatozoa. Before detection or discrimination the sperm may be oriented hydrodynamically and the sperm may be separated into individual droplets that then may be electrically charged. After detection or discrimination, the sperm are sorted by electric field—charged droplet interactions.